Song Chang-Zheng, Wang Qing-Wei, Song Chang-Cheng, Bai Zeng-Liang
Laboratory of Immunobiology, College of Life Sciences, Shandong University, Jinan 250100, Shandong Province, China.
World J Gastroenterol. 2004 Feb 1;10(3):389-92. doi: 10.3748/wjg.v10.i3.389.
A strategy for viral vaccine design is the use of conserved peptides to overcome the problem of sequence diversity. At present it is still unclear whether conserved peptide is safe as a candidate vaccine. We reported it here for the first time not only to highlight the biohazard issue and safety importance for viral peptide vaccine, but also to explore the effect of a fully conserved peptide on HBV replication within the carboxyl terminus of HBx.
We synthesized the fully conserved peptide (CP) with nine residues, FVLGGCRHK. HBV-producing 2.2.15 cells were treated with or without 3.5 microM CP for 36 hours. Quantitative detection of viral DNA was performed by real-time PCR. HBV antigens were determined by enzyme-linked immunoadsorbent assay (ELISA). Quantitative analyses of p53 and Bax proteins were based on immunofluorescence. Flow cytometry was performed to detect cell cycle and apoptosis.
Both extracellular and intracellular copies of HBV DNA per ml were significantly increased after incubation with 3.5 microM of CP. HBsAg and HBeAg in the cultured medium of CP-treatment cells were as abundant as untreated control cells. CP influenced negatively the extracellular viral gene products, and 3.5 microM CP could significantly inhibit intracellular HBsAg expression. In response to CP, intracellular HBeAg displayed an opposite pattern to that of HBsAg, and 3.5 microM CP could efficiently increase the level of intracellular HBeAg. Flow cytometric analyses exhibited no significant changes on cell cycle, apoptosis, p53 and Bax proteins in 2.2.15 cells with or without CP.
Together with the results generated from the synthetic peptide, we address that the conserved region, a domain of HBx, may be responsible for modulating HBV replication. As conserved peptides from infectious microbes are used as immunogens to elicit immune responses, their latent biological hazard for human beings should be evaluated.
病毒疫苗设计的一种策略是使用保守肽来克服序列多样性问题。目前，保守肽作为候选疫苗是否安全仍不清楚。我们首次在此报道，不仅是为了强调病毒肽疫苗的生物危害问题和安全重要性，也是为了探索一个完全保守的肽对HBx羧基末端内乙肝病毒复制的影响。
我们合成了具有九个残基FVLGGCRHK的完全保守肽（CP）。用或不用3.5微摩尔CP处理产生乙肝病毒的2.2.15细胞36小时。通过实时聚合酶链反应进行病毒DNA的定量检测。通过酶联免疫吸附测定（ELISA）测定乙肝病毒抗原。基于免疫荧光对p53和Bax蛋白进行定量分析。进行流式细胞术检测细胞周期和凋亡。
与3.5微摩尔CP孵育后，每毫升乙肝病毒DNA的细胞外和细胞内拷贝数均显著增加。CP处理细胞培养基中的乙肝表面抗原（HBsAg）和乙肝e抗原（HBeAg）与未处理的对照细胞一样丰富。CP对细胞外病毒基因产物有负面影响，3.5微摩尔CP可显著抑制细胞内HBsAg表达。响应CP时，细胞内HBeAg呈现与HBsAg相反的模式，3.5微摩尔CP可有效提高细胞内HBeAg水平。流式细胞术分析显示，有无CP的2.2.15细胞在细胞周期、凋亡、p53和Bax蛋白方面无显著变化。
结合合成肽产生的结果，我们指出HBx的一个结构域即保守区域可能负责调节乙肝病毒复制。由于来自传染性微生物的保守肽被用作免疫原以引发免疫反应，因此应评估它们对人类潜在的生物危害。
